﻿Thrixspermumtaeniophyllum (Orchidaceae, Epidendroideae), a new species from southwest China, based on molecular and morphological evidence

﻿Abstract Thrixspermumtaeniophyllum is described as a new orchid species from Wenchuan County, Sichuan Province of southwest China. It is morphologically similar to T.japonicum, but it differs from the latter in having branched stems, slightly fleshy strap-shaped leaves, longer inflorescences with 3–6 flowers and a capitate gynandrium with a lip-shaped mouth opening. Its species status is also supported by molecular phylogenetic analyses, based on nuclear ribosome internal transcribed spacer (nrITS) and three chloroplast DNA fragments (matK, psbA-trnH and trnL-F), which showed distinct systematic boundaries from the most morphologically similar T.japonicum and their morphological relatives T.saruwatarii and T.pygmaeum.


Introduction
Thrixspermum Lour. (1790) is a genus of mostly medium-sized epiphytes and lithophytes in the family Orchidaceae Juss. and it is known to include ca. 160 species widely distributed from tropical and subtropical Asia to the islands of the western Pacific islands (Chen et al. 2009;Chase et al. 2015;Kumar et al. 2017). This genus is characterised by the persistent floral bracts, a three-lobed labellum and the four waxy subglobose pollinia grouped into two unequal masses in appearance (Loureiro 1790;Chen et al. 2009). It is also a congregation of elusive orchids with limited floral materials for morphological comparison due to their rather short flowering period (Govaerts et al. 2016). From accounts of ca. 17 species distributed in southern China, only one species, T. japonicum (Miq.) Reichenbach fils (1878), has been recorded from Sichuan Province (Song et al. 2009;Kumar et al. 2017;Zhou et al. 2021).
As part of a continuous inventory of orchids from Sichuan, China, we have conducted continuous field explorations in the Wenchuan section of the Giant Panda National Park. During a field trip in March 2022, we encountered an interesting epiphytic orchid in Wolong National Nature Reserve (Wenchuan County, Sichuan Province, China), that we had initially identified it as Thrixspermum japonicum, based on its pendulous inflorescence, golden-yellow flowers and orange-red striped lateral lobes of the labellum. However, upon a critical morphological observation and comparison with available specimens including the type materials of two of the three [we were unsuccessful to trace the type specimen(s) of T. pygmaeum (King & Pantl.) Holttum (1960)] morphologically related species (Fig. 1A-I), for example, T. japonicum (P. F. V. Siebold, s.n., L) and T. saruwatarii (Hayata) Schlechter (1919) (T01201, TI), we assumed it to be a new species of Thrixspermum that we are now describing hereafter.

Morphological observations
Morphological information (including the colour, size and shape of the roots, stems, leaves, flowers and floral parts, details see Table 1 Characters of the last three species are modified from the respective protologues (Miquel 1866;King and Pantling 1898;Hayata 1916) and Flora of China (Chen et al. 2009 Voucher information for the four specimens used for morphological observations and their collection location are detailed in the taxonomic treatment. The terminology in Beentje (2012) was followed for the description.

DNA extraction and sequencing
The sequences of four individuals of this new species from two different areas (Wolong and Gengda towns) in Wenchuan County and two individuals of

T. japonicum (vouchers Jun-Yi Zhang & Yue-Hong Cheng ZJY187 and Jun-Yi
Zhang & Yue-Hong Cheng ZJY188, deposited at CDBI) were newly obtained in this study with the following protocols. Total DNA was extracted from silica-gel dried leaves via a Plant DNA Isolation Kit Foregene,Chengdu,China). The sequences were amplified by means of the primers (Table 2) used in previous studies of Thrixspermum (Li et al. 2014;Zou et al. 2015

Phylogenetic analyses
A total of 54 accessions representing 44 taxa were incorporated in the phylogenetic analysis, including Phalaenopsis marriottiana (Rchb. f.) Kocyan & Schuiteman (2014) as outgroup. The ingroup includes 36 entities of Thrixspermum representing 26 species and 17 taxa belonging to six related genera in Aeridinae (Orchidaceae, Epidendroideae) following the two previous studies of Li et al. (2014) and Zou et al. (2015). The detailed information concerning the sampled taxa, voucher specimens and GenBank accession numbers (including the sequences retrieved from GenBank) used for the phylogenetic analyses are summarised in Appendix 1. The nrITS, matK, psbA-trnH and trnL-F matrices contain 40, 22, 13 and 13 taxa, respectively (Appendix 1). All sequences were aligned using MAFFT v.7.475 (Katoh and Standley 2013) with default parameters. The incongruence length difference test (ILD) was used to quantify the conflicts between nuclear DNA (nrITS) and plastid DNA (matK, psbA-trnH, trnL-F) data in PAUP v.4.0a169 (Darlu and Lecointre 2002;Swofford 2002). The ILD Test (P = 0.11) indicated that nrITS and plastid datasets were suitable for combined analysis in Thrixspermum and, thus, the results are based on the combined data of nrITS and three plastid markers. The nucleotide substitution models for these data matrices were estimated using the software jModelTest v.2.1.6 (Posada 2008) and the best fit models were selected using the corrected Akaike Information Criterion (AICc). Bayesian Inference (BI) and Maximum Likelihood (ML) analyses were performed to infer the phylogenetic relationships within the combined dataset. The BI analysis was conducted using MrBayes v.3.2.7a (Ronquist and Huelsenbeck 2003), with two separate Markov Chain Monte Carlo (MCMC) chains (1,000,000 generations and sampled every 1,000 generations). The first 25% of the trees were discarded as burn-in and the remaining trees were used to generate a majority-rule consensus tree. The ML analysis was performed using IQ-TREE v.1.4.2 (Nguyen et al. 2014) with branch support estimated using 2,000 replicates of both SH-like approximate likelihood-ratio test (SH-aLRT) (Guindon et al. 2010) and the ultrafast bootstrapping algorithm (UFboot) (Minh et al. 2013).

Results
The aligned nrITS matrix of 48 accessions (40 taxa (Table 2). Phylogenetic analyses indicated that the 26 included taxa of Thrixspermum formed a well-supported monophyletic group (Fig. 2). Four individuals of the inferred new species from the two sites in Wenchuan County were resolved as a strongly-supported monophyletic lineage ( Fig. 2; BI/ML = 1/100%), which further clustered with T. japonicum, T. saruwatarii and T. pygmaeum into a subclade ( Fig. 2; BI/ML = 1/100%). These four species also showed certain morphological similarities (referring to Fig. 1 and Table 1). It is noted that T. japonicum is the most related species to the novelty by sharing with the new species pendulous inflorescence, inside brownish-striped lateral lobes and densely hairy small triangular mid-lobe of labellum (Fig. 1B, E). Besides, the following morphological diagnosis, their molecular boundary is clearly shown by the positions of their respective individuals as well (Fig. 2).

Diagnosis.
It is most similar to Thrixspermum japonicum in morphology, but it differs from the latter by its often-branched stems (vs. unbranched stems), slightly fleshy strap-shaped leaves 5-7 cm long (vs. thinly leathery oblong leaves 2-4 cm long), longer inflorescences with 3-6 flowers (vs. shorter inflorescences with 2-3 flowers) and its capitate gynandrium with a lip-shaped mouth opening (vs. conical gynandrium with a triangular mouth opening). It also resembles T. saruwatarii and T. pygmaeum and morphological comparison amongst the four species is visualised in Fig. 1 and summarised in Table 1.
Distribution and habitat. Thrixspermum taeniophyllum was found in Wenchuan County, Sichuan Province, southwest China. It is epiphytic on trees in coniferous and broadleaf mixed forest at an elevational range between 1260 and 1770 m.
Phenology. Flowering in March and April. Etymology. The specific epithet taeniophyllum is a compound adjective referring to the shape of leaves of this new species. A Chinese name, dai ye bai dian lan (带叶白点兰), is also suggested, based on the its leaf feature.
Additional specimens examined.

Conflict of interest
The authors have declared that no competing interests exist.

Ethical statement
No ethical statement was reported. and BX revised the manuscript. All authors read and approved last manuscript.

Data availability
All of the data that support the findings of this study are available in the main text or Appendix 1.